PAPER1

Cytotoxic and Inflammatory Responses Induced by Outer Membrane
Vesicle-Associated Biologically Active Proteases from Vibrio cholerae
Ayan Mondal,a
Rima Tapader,a
Nabendu Sekhar Chatterjee,b
Amit Ghosh,a
* Ritam Sinha,c
Hemanta Koley,c
Dhira Rani Saha,d
Manoj K. Chakrabarti,a
Sun Nyunt Wai,e
Amit Pala
Division of Pathophysiology,a
Division of Biochemistry,b
Division of Bacteriology,c
and Histology and Electron Microscopy,d
National Institute of Cholera and Enteric
Diseases, Kolkata, India; Department of Molecular Biology, Umea Centre of Microbial Research, Umea University, Umea, Swedene
Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemaggluti-
nin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our
present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and
transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in
Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemor-
rhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which
were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in
intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biolog-
ically active proteases which may play a role in cytotoxic and inflammatory responses.
Vibrio cholerae is the causative agent of the life-threatening dis-
ease cholera. Cholera epidemics in Haiti in 2010 provide evi-
dence that it still remains an ongoing public health threat (1).
Strains of the El Tor biotype O1 serogroup are responsible for
seventh pandemic and recent cholera outbreaks (2). Cholera toxin
(CT) and toxin-coregulated pilus (TCP) have been identified as
the major virulence factors for V. cholerae pathogenesis. CT is
responsible for profuse watery diarrhea, whereas TCP is essential
for sustaining colonization of human small intestine. V. cholerae
also secretes several proteases which may also play a role in its
pathogenesis (3).
The major protease secreted by V. cholerae strains is the 35-kDa
hemagglutinin protease (HAP) (4). HAP has been reported to
accelerate the bacterial detachment from cultured cells by diges-
tion of V. cholerae adhesins (5). HAP modulates the enterotoxige-
nicity of cholera toxin by nicking the A subunit of CT (6). HAP
also plays a role in processing hemolysin to its mature form by
removal of a 15-kDa N-terminal peptide (7). As shown in the
above-mentioned studies, HAP plays an indirect role in V. chol-
erae pathogenesis. The possibility of its direct role in pathogenesis
has been shown by Ghosh et al. (8). They reported that purified
HAP from a V. cholerae non-O1, non-O139 strain showed a hem-
orrhagic response in rabbit ileal loops (RILs) and an increase in
intestinal short-circuit current in Ussing’s chamber (8). Besides
HAP, the other major well-characterized metalloprotease is a 97-
kDa Vibrio cholerae protease, PrtV which has been shown to play a
role in virulence in the Caenorhabditis elegans infection model (9).
In an earlier study, we also reported the presence of a novel serine
protease encoded by VC1649 (VesC) in a hapA prtV mutant Vibrio
cholerae O1 strain and showed its role in the hemorrhagic re-
sponse in the rabbit ileal loop model (10).
Gram-negative bacteria, including V. cholerae, use 6 different
secretion systems (type I to type VI) to transport important viru-
lence factors to the cell envelope and the extracellular milieu (11).
Outer membrane vesicles (OMVs) may also serve as a mechanism
of delivering active virulence factors into host cells. The virulence
factors are protected from digestion by host proteases and are
secreted not individually but as multiple factors delivered in a
discrete package (12). Toxin delivery mediated by OMVs is recog-
nized as a potent virulence mechanism for many bacterial patho-
gens. Association of toxins with OMVs have been reported for
many pathogenic Gram-negative bacteria. Toxins such as ClyA
cytotoxin, ␣-hemolysin, and CNF1 of Escherichia coli, cytolethal
distending toxin (CDT) of Campylobacter jejuni, and vacuolating
cytotoxin A (VacA) of Helicobacter pylori are secreted through
OMVs (13, 14, 15, 16, 17). Bomberger et al. showed that multiple
virulence factors, such as ␤-lactamase, alkaline phosphatase, he-
molytic phospholipase C, and Cif, are directly delivered into the
host cytoplasm via fusion of OMVs with lipid rafts in the host
plasma membrane and that these toxins are rapidly distributed to
specific subcellular locations to affect host cell biology (18).
OMV-mediated secretion of biologically active CT, Vibrio chol-
erae cytolysin (VCC), and PrtV has been reported in V. cholerae
(19, 20, 21).
Recent studies on proteomic analysis of V. cholerae OMVs have
the shown presence of both HAP and the 59-kDa serine protease
VesC (22). However, the functions of these vesicle-associated pro-
teases have not been elucidated. Our study shows that V. cholerae
OMVs secrete biologically active proteases which may play a role
in cytotoxic and inflammatory responses.
Received 5 November 2015 Returned for modification 23 December 2015
Accepted 20 February 2016
Accepted manuscript posted online 29 February 2016
Citation Mondal A, Tapader R, Chatterjee NS, Ghosh A, Sinha R, Koley H, Saha DR,
Chakrabarti MK, Wai SN, Pal A. 2016. Cytotoxic and inflammatory responses
induced by outer membrane vesicle-associated biologically active proteases from
Vibrio cholerae. Infect Immun 84:1478–1490. doi:10.1128/IAI.01365-15.
Editor: A. Camilli
Address correspondence to Amit Pal, apal7364@rediffmail.com.
* Present address: Amit Ghosh, Department of Physiology, All India Institute of
Medical Sciences, Bhubaneswar, India.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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MATERIALS AND METHODS
Ethics statement. Animal experiments were performed after obtaining
necessary permission from the Institutional Animal Ethical Committee
(IAEC). The IAEC/CPCSEA approval number is APRO 7624/11/2010.
Bacterial strains. The bacterial strains, and oligonucleotides used in
this study are listed in Table 1. V. cholerae cells were routinely grown in
tryptic soy broth (TSB) (Difco, USA) at 37°C with shaking as described
earlier (10). E. coli TOP 10 cells were grown overnight in Luria broth (LB)
at 37°C with shaking at 200 rpm. Antibiotics were used at the following
concentrations: streptomycin, 100 ␮g/ml; kanamycin, 40 ␮g/ml; and am-
picillin, 100 ␮g/ml. Bacterial cells, including mutants, were maintained at
Ϫ70°C in LB containing 20% sterile glycerol.
Complementation of the VC1649 gene in V. cholerae strain CHA6.8
⌬prtV ⌬VC1649. The VC1649 gene was amplified from genomic DNA
isolated from CHA6.8 ⌬prtV using VesC forward and reverse primers
(Table 1). Cloning was performed as described by Gosink et al. (23). The
amplified VC1649 gene was cloned into the pBAD-TOPO TA (Invitrogen,
USA) cloning vector under the control of an arabinose-inducible pro-
moter using a cloning kit from Invitrogen and was transformed in E. coli
TOP 10 cells. Transformed colonies (Ampϩ
) were selected in a Luria agar
(LA) plate containing 100 ␮g/ml ampicillin. Positive clones were selected
from the transformed colonies by colony PCR with the pBAD forward and
VesC reverse primers supplied in the cloning kit, and plasmids were iso-
lated using a plasmid isolation kit (Thermo Fisher, USA). Plasmid
pVC1649 was transformed into V. cholerae strain CHA6.8 ⌬prtV
⌬VC1649 by electroporation (23).
IsolationandpurificationofOMVsfromV.choleraestrains.V.chol-
erae cells were grown in 2 liter of TSB for 18 h at 37°C under shaking
conditions, and outer membrane vesicles (OMVs) were prepared from
late-log-phase culture as described previously (17). Crude vesicles were
isolated from strain Comp VC1649 grown in 250 ml of TSB using L-ara-
binose induction as described by Chen et al. (24). Isolated crude OMVs
from C6709, OMVs from its isogenic protease mutants, and OMVs from
Comp VC1649 were purified by density gradient centrifugation on a dis-
continuous Optiprep-iodixanol (Sigma, USA) gradient as described by
Elluri et al. (20). Fractions of equal volumes were sequentially removed
after density gradient centrifugation and analyzed by Western blotting
using anti-OmpU antibody. OmpU is the marker protein for V. cholerae
OMVs. The protein content of purified OMVs was estimated using the
Bradford assay.
Transmission electron microscopy (TEM). Twenty micrograms of
purified OMVs was placed on carbon-coated nickel grids (300 mesh)
(Sigma, USA), stained with 2% aqueous uranyl acetate, rinsed, and exam-
ined with a transmission electron microscope operating at 60 kV (FEI
Tecnai 12; Bio, Twin, The Netherlands).
RaisingofantiseraagainstHAP,VesC,andOMVs.HAPwaspurified
from the culture supernatant of C6709 as described by Ghosh et al. (8).
Antiserum to purified HAP was prepared by immunizing a New Zealand
White rabbit by intramuscular injection with 100 ␮g of HAP emulsified
with an equal volume of Freund’s complete adjuvant (Sigma, USA). This
was followed by four booster injections with 100 ␮g of HAP and incom-
plete adjuvant (Sigma, USA) at 7-day intervals. Blood samples were col-
lected from rabbits on day 0 and 3 days after the final injections and were
allowed to clot at room temperature for 30 min. Sera were collected,
followed by centrifugation (1,000 rpm, 10 min), dilution in phosphate-
buffered saline (PBS) (Sigma, USA), and storage at Ϫ80°C (25). Antise-
rum against the 59-kDa serine protease (VesC) was also raised in a New
Zealand White rabbit. VesC was partially purified as described earlier
(10). The mass spectrometrically identified band of VesC in a native poly-
acrylamide gel was excised, homogenized in PBS, and administered into
an intramuscular region of a New Zealand White rabbit. Antiserum
against VesC was drawn from the rabbit after four booster injections as
described above. Antiserum against OMVs was prepared by immunizing
7-week-old mice (BALB/c). Mice were immunized subcutaneously with
50 ␮g of OMVs isolated from C6709 at the first, second, and third weeks,
followed by a booster dose of 75 ␮g of the OMVs at the fourth week. The
serum was collected, and the antibody titer was determined by enzyme-
linked immunosorbent assay (ELISA) with the preimmune serum as a
control.
SDS-PAGE and Western blot analysis. V. cholerae OMVs were sepa-
rated by 10% SDS-PAGE. Briefly, 20 ␮g each of purified OMVs and
whole-cell lysates of C6709 and CHA6.8 were mixed with Laemmli sample
TABLE 1 Bacterial strains, plasmids, and primers
Strain, plasmid, or primer Relevant characteristics
Source or
reference
Vibrio cholerae strains
C6709 Wild type (O1 El Tor); Smr
56
CHA6.8 C6709 ⌬hapA::Kan; Smr
Kmr
10
CHA6.8 ⌬prtV CHA6.8 ⌬prtV; Smr
Kmr
10
CHA6.8 ⌬prtV ⌬VC1649 (VesC mutant) CHA6.8 ⌬prtV ⌬VC1649; Smr
Kmr
10
Comp VC1649 CHA6.8 ⌬prtV ⌬VC1649::pVC1649; Smr
Kmr
Ampr
This study
E. coli strain TOP 10 FϪ
mcrA ⌬(mrr-hsdRMS-mcrBC) ␾80lacZ⌬M15⌬lacX74 recA1
araD139 ⌬(ara-leu)7697 galU galK rpsL (Strr
) endA1 nupG
Invitrogen, USA
Plasmids
pBAD TOPO-TA Ampϩ
TA cloning vector plasmid Invitrogen, USA
pVC1649 pBAD TOPO with Vibrio cholerae VC1649 (VesC) This study
Primers
IL 8 F1 5=-ATGGGGAAGGTGAAGGTCGG-3= 57
IL 8 R1 5=-TCTCAGCCCTCTTCAAAAACTTCTC-3= 57
GAPDH F1 5=-ATGGGGAAGGTGAAGGTCGG-3= 58
GAPDH R1 5=-GGATGCTAAGCAGTTGGT-3= 58
pBAD F 5=-ATGCCATAGCATTTTTATCC-3= Invitrogen, USA
pBAD R 5=-GATTTAATCTGTATCAGG-3= Invitrogen, USA
VesC F 5=-ATGAACAAAACATTTTTATC-3= This study
VesC R 5=-TCAGACGCGTTGACGACG-3= This study
OMV-Associated Active Proteases in V. cholerae
May 2016 Volume 84 Number 5 iai.asm.org 1479Infection and Immunity

buffer (1:1) and boiled for 10 min prior to electrophoresis. Proteins with
known molecular weights (Thermo Fisher, USA) were used as markers.
Gels were stained with Coomassie brilliant blue stain after electrophoresis.
For immunoblotting, the separated proteins were transferred onto a ni-
trocellulose membrane (Bio-Rad, USA) by the wet transfer method using
a Trans-Blot apparatus (Bio-Rad, USA). The membrane was blocked at
room temperature for 1 h in 5% nonfat dry milk powder (Bio-Rad, USA)–
PBS–Tween 20. After incubation, the membrane was washed and incu-
bated with rabbit anti-HAP (1:1,000 dilution in 5% [wt/vol] nonfat dry
milk), and anti-VesC (1:10,000 dilution in 5% [wt/vol] nonfat dry milk)
antisera, followed by incubation for 1 h at room temperature with a
1:10,000 dilution of anti-rabbit horseradish peroxidase-conjugated sec-
ondary antibody (Thermo Fisher, USA). The blots were visualized with
ECL Western blotting detection reagent (Millipore, USA).
Proteolytic digestion of prohemolysin. Proteolytic digestion of El
Tor prohemolysin was performed as described earlier (7). Briefly, 10 ␮l of
79-kDa prohemolysin (1 mg/ml) was mixed with 10 ␮l (100 ␮g/ml) of
purified C6709 OMVs and incubated at 37°C for 30 min, and SDS-PAGE
was performed. Untreated purified prohemolysin and HAP-treated pro-
hemolysin were used as negative and positive controls, respectively.
Proteinase K susceptibility assay. The proteinase K susceptibility as-
say was performed as described previously (26). Briefly, OMVs were
treated with proteinase K (1 ␮g/ml) for 30 min at 37°C in either the
presence or absence of 1% SDS, and samples were analyzed by immuno-
blotting against anti-HAP and anti-VesC antibodies raised in rabbit.
Media for cell lines and culture conditions. Int407 and T84 human
intestinal epithelial cells were used in this study. Int407 cells were grown in
25-ml tissue culture flasks (BD Biosciences) containing minimal essential
medium (MEM) (Gibco, USA) supplemented with 10% fetal bovine se-
rum (Gibco, USA) along with penicillin G and streptomycin sulfate
(Sigma, USA) at 37°C in a CO2 incubator (Heraeus, Germany). Cells at 80
to 90% confluence were seeded in 6-well and 24-well tissue culture plates
(BD Biosciences) at a concentration of approximately 105
cells/well. Cells
were permitted to reach confluence by allowing them to grow for 24 h in
a CO2 incubator. T84 cells were used for interleukin-8 (IL-8) assay, and
the cells were maintained in Dulbecco’s modified Eagle’s Medium-nutri-
ent F12 (DMEM-F12) (Gibco, USA) supplemented with antibiotic solu-
tion as described above.
Tissue culture assay. The tissue culture assay was performed as de-
scribed by Ghosh et al. (8). Briefly, OMVs from C6709 and its isogenic
knockout strains were filter sterilized and serially diluted in serum-free
MEM from 0.5 ␮g/ml to 100 ␮g/ml. The exhaust media were aspirated
from 24-well tissue culture plates containing Int407 cells grown to con-
fluence and washed with PBS, and serially diluted OMVs were added and
grown at 37°C in a CO2 incubator for 24 h. Purified HAP was used as
positive control. Results were analyzed by phase-contrast microscopy and
photographs taken (CK 40; Olympus).
OMV internalization assay. Int407 cells were incubated with 20
␮g/ml of purified OMVs from C6709 and its isogenic protease mutants
for 1 h at 37°C in a 5% CO2 atmospheric chamber to monitor internaliza-
tion of V. cholerae OMVs. The effects of several endocytic inhibitors (nys-
tatin, dynasore, and methyl-␤-cyclodextrin [M␤CD]) on OMV uptake
were examined by pretreating Int407 cells with nystatin (25 ␮g/ml) and
dynasore (80 ␮M) for 1 h and with M␤CD (1 mg/ml) (Sigma, USA) for 30
min as described by Parker et al. (27). This was followed by incubation
with purified OMVs for 1 h. Cells were then fixed with 4% paraformalde-
hyde at 4°C for 20 min, followed by permeabilization with 1% Triton
X-100 in PBS for 20 min at room temperature. Mouse antiserum against
OMVs (1:2,000) was added to OMV-treated cells and incubated overnight
at 4°C in a moist chamber. Cells were washed with sterile PBS and incu-
bated with tetramethyl rhodamine isocyanate (TRITC)-conjugated anti-
mouse secondary antibody (1:500) and were kept in the dark at 37°C for
90 min.
For colocalization studies, primary antibodies against HAP (1:1,000)
and OMVs (1:2,000) raised in rabbit and mouse, respectively, were added
to C6709 and CHA6.8 OMV-treated cells. Primary antibodies against
VesC (1:2,500) and OMVs (1:2,000) were added to cells treated with
OMVs from C6709, isogenic protease mutants of C6709, and the VC1649
complemented strain and incubated overnight as described above. Cells
were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-
rabbit IgG (1:500) and TRITC-conjugated anti-mouse antibody (1:500)
and were kept in the dark at 37°C for 90 min after incubation. Cells were
washed and resuspended in 100 ␮l of PBS. Approximately 20 ␮l of resus-
pended sample was used to make a thin film on a glass slide. One drop of
90% glycerol was added, a thin glass coverslip was placed on it, and the
slide was viewed under a confocal microscope (model LSM 510 META;
Zeiss, Germany) and excited at 488 nm and 543 nm for FITC and TRITC,
respectively. The emission filters used were 475 nm to 525 nm for FITC
and 558 nm to 601 nm for TRITC. Experiments were also done with PBS
as negative control. Merged images were developed using Image J soft-
ware.
Mouse intestinal fluid accumulation assay. The mouse intestinal
fluid accumulation assay was performed as described earlier (28). Briefly,
20 ␮g of purified OMVs from V. cholerae and its isogenic protease mutant
strains was injected into ligated intestinal segments of about 4 cm in
length from 4- to 5-week-old adult BALB/c mice. All experimental proce-
dures were performed under anesthesia with ketamine. After 6 h, mice
were sacrificed and loops were excised. The enterotoxic activity was de-
termined by measuring the fluid accumulation (FA) ratio, which is the
ratio of fluid accumulated in the intestinal loop to the length of the loop
(g/cm). PBS was used as a negative control. Values were expressed as
mean Ϯ standard deviation (SD) of the mean (n ϭ 5 mice per group).
Histopathological studies of mouse ileal loops (MILs). Mouse ileal
tissues were collected and fixed in 10% neutral buffered formalin solution
for histopathological studies. The tissues were further embedded in par-
affin and processed following the standard protocol. Three- to four-mi-
crometer thin sections were cut in a microtomy rotor (Leica, Germany).
The sections were strained with hematoxylin and eosin and observed un-
der a light microscope. Photographs were taken under a magnification of
ϫ40 with a Leica DMLB microscope (Solms, Germany) equipped with a
digital imaging system.
Flow cytometric analysis. Flow cytometric analysis was performed as
described by Thay et al. (29). Briefly, Int 407 cells were treated with OMVs
from V. cholerae strain C6709 and its protease knockout mutant strains at
concentrations of 20 ␮g/ml and 40 ␮g/ml for 4 h and then stained with
fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium
iodide (PI) (BD Pharmingen) according to the manufacturer’s instruc-
tions. The cells were stained with FITC-annexin V in annexin V binding
buffer for 10 min, followed by addition of PI, and analyzed by flow cy-
tometry using Cell Quest Pro software (BD Biosciences). A total of 104
cells were acquired for data analysis in each set of experiments. Values
were expressed as mean Ϯ SD of the mean for 3 different observations
(n ϭ 3).
CT-bead ELISA. CT concentrations in OMVs and culture superna-
tants of C6709 and its isogenic protease mutants grown in TSB were mea-
sured by bead ELISA as described by Oku et al. (30). Culture supernatant
of V. cholerae N16961 grown in Syncase medium was used as a positive
control.
Mouse intestinal colonization assay. Pathogen-free adult 4- to
5-week-old BALB/c mice were used for the colonization assay. Prior to the
experiment, all mice were fasted for 24 h with free access to sterile water.
The experiment was performed as described by Olivier et al. (31). Briefly,
mice were injected intraperitoneally with a mixture of 35 mg/kg ketamine
and 5 mg/kg xylazine for anesthesia. Two hundred microliters of suspen-
sions of ϳ109
viable bacterial cells was administered orally with two bo-
luses of 0.5 ml of 5% (wt/vol) NaHCO3. All mice were kept in microiso-
lator cages with free access to food and sterile water after inoculation. Mice
were euthanized with Euthanasia 6 solution after 18 h of oral inoculation.
The small intestine above the cecum was collected after dissection, ho-
mogenized, and then serially diluted in sterile PBS (pH 7.4). Homoge-
Mondal et al.

nized materials were plated on appropriate antibiotic-containing tryptic
soy agar (TSA) plates and incubated for 24 h. All experiments were per-
formed in triplicates, and values were expressed as mean Ϯ SD (n ϭ 10
mice per group) (*, P Ͻ 0.05; **, P Ͻ 0.01) and compared with the CFU
obtained for C6709.
ELISA and semiquantitative RT-PCR for IL-8 estimation. For en-
zyme-linked immunosorbent assays (ELISAs), the growth medium was
removed from the plates. Cells were washed with PBS (Sigma, USA) and
maintained in antibiotic- and serum-free DMEM-F12. The cells were
treated with 10 ␮g/ml of V. cholerae OMVs from C6709 and its isogenic
knockout mutant strains for IL-8 stimulation. Supernatants were col-
lected after 24 h and centrifuged to remove dead cells, and IL-8 concen-
trations were determined by ELISA using an ELISA kit from Invitrogen
(USA) according to the instructions of the manufacturer. For reverse
transcription-PCR (RT-PCR), total RNA was isolated from the OMV-
treated T84 cells using TRIzol reagent (Invitrogen, USA). RNA was quan-
tified by spectrophotometric analysis and the quality of the RNA samples
determined by estimating the A260/A280 ratio. cDNA was prepared from
1.5 ␮g of total RNA using the Superscript II first-strand synthesis system
for RT-PCR (Invitrogen, USA). A 2.5-␮l portion of cDNA was PCR am-
plified in a 25-␮l reaction volume containing 10 mM Tris-Cl (pH 8.3), 50
mM KCl, 2.0 mM MgCl2, 2.5 mM each deoxynucleoside triphosphate
(dNTP), 20 pmol of each primer, and 1 U of Taq DNA polymerase
(Roche, Germany). The primers used for IL-8 and GAPDH (glyceralde-
hyde-3-phosphate dehydrogenase) are listed in Table 1. The PCR condi-
tions were as follows: an initial denaturation step at 95°C for 5 min fol-
lowed by 35 cycles of 95°C (1 min), 55°C and 60°C for IL-8 and GAPDH,
respectively (1 min), and 72°C (30 s). The final extension step was done at
72°C for 7 min. The products were electrophoresed on a 2% agarose gel,
stained with ethidium bromide, and visualized using a gel documentation
system (Bio-Rad, USA). The data from ELISA and RT-PCR was recorded
as mean Ϯ SD from at least three groups of experiments. Densitometric
quantification of RT-PCR bands was performed with Quantity One soft-
ware (Bio-Rad, USA).
Statistical analysis. All experiments were performed in triplicates.
Values were expressed as mean Ϯ standard deviation of the mean. Vari-
ables were compared for significance using two-way analysis of variance
and the Bonferroni test, with one asterisk indicating a P value between
0.01 to 0.05 and two asterisks indicating a P value between 0.01 to 0.001.
RESULTS
Fractions of HAP and VesC are associated with OMVs of Vibrio
cholerae C6709 and hapA mutant strains, respectively. V. chol-
erae secretes abundant vesicles during its normal course of growth
(32). To investigate the association of proteases with outer mem-
brane vesicles (OMVs), crude OMVs were isolated from culture
supernatants of the pathogenic O1 El Tor strain C6709 and its
protease mutants by ultracentrifugation, followed by purification
using Optiprep density gradient centrifugation. Fractions ob-
tained from the gradient centrifugation of OMVs were analyzed
by immunoblotting using OmpU antiserum. Fractions showing
reactivity with OmpU antiserum were examined by transmission
electron microscopy for ultrastructural analysis and used for fur-
ther experiments. OMVs of C6709 appeared as spherical electron-
dense materials of heterogeneous sizes with a diameter in the
range of 25 to 150 nm. Major morphological differences were not
observed in OMVs from the isogenic protease mutant strains and
OMVs from Comp VC1649. To investigate OMV-associated se-
cretion of HAP, OMVs from HAP-producing C6709 and ⌬hapA
strain CHA6.8 were subjected to SDS-PAGE along with whole-cell
lysates from the respective strains, and immunoblotting was per-
formed using polyclonal antiserum against HAP. Concentrated
OMV-free culture supernatants from C6709 and purified HAP
were used as positive controls. An immunoreactive band corre-
sponding to HAP was observed only in C6709 OMVs. The pres-
ence of HAP was not observed in whole-cell lysate prepared from
strain C6709. The immunoblot results showed the presence of the
mature 45-kDa form of HAP in OMVs instead of its processed
35-kDa form, which was present in OMV-free culture supernatant
of C6709 (Fig. 1B). The presence of VesC in the OMVs was de-
tected by immunoblotting using VesC antiserum (1:10,000). VesC
was detected in the OMVs from the CHA6.8 and CHA6.8 ⌬prtV
strains and the VC1649 complemented construct. In contrast, its
presence was not observed in OMVs from C6709 and CHA6.8
⌬prtV ⌬VC1649 (Fig. 1C). The above results demonstrated that
FIG 1 Association of HAP and VesC with V. cholerae OMVs by immunoblot analysis. (A) Ten percent SDS-PAGE profile of the OMVs of Vibrio cholerae strains
C6709 (lane 3), CHA6.8 (lane 4), CHA6.8 ⌬prtV (lane 5), CHA6.8 ⌬prtV ⌬VC1649 (lane 6), and Comp VC1649 (complemented) (lane 7). Five micrograms of
purified HAP was resolved in lane 2, and a prestained protein ladder (Thermo Fisher, USA) is in lane 1. (B) Immunoblot analyses with OMVs from C6709 and
CHA6.8 and whole-cell lysates from the respective strains against HAP antiserum. Purified 45-kDa and 35-kDa HAP and concentrated OMV-free culture
supernatant of C6709 were used as positive controls. (C) Immunoblot analyses with OMVs of C6709 and its isogenic protease mutants against VesC antiserum.

HAP is secreted in association with OMVs from the V. cholerae
wild-type strain C6709, whereas the serine protease VesC is se-
creted in association with OMVs from ⌬hapA strains.
LocalizationofproteasewithintheOMVs.Localizationofthe
HAP and VesC within the OMVs was confirmed by proteinase K
susceptibility assay. Clear proteolytic digestion of HAP was ob-
served only when OMVs from C6709 were incubated with protei-
nase K in the presence of 1% SDS, and this proteolytic digestion
was inhibited in the presence of phenylmethylsulfonyl fluoride
(PMSF) (Fig. 2A). A similar result was observed with OMV-asso-
ciated VesC in the CHA6.8 ⌬prtV strain (Fig. 2B). These results
confirmed the luminal localization of HAP and VesC within the
vesicles.
Protease-associated OMVs bind to and are internalized by
human intestinal epithelial cells in a protease-independent
manner. The interaction of OMVs with Int407 cells was studied
by confocal microscopy. OMV-specific red fluorescence was ob-
served throughout Int407 cells treated with OMVs from C6709
and its isogenic protease mutant strains (Fig. 3A, panels 4 and 7,
and C, panels 1, 4, 7, and 10). In contrast, no fluorescence was
observed in cells treated with buffer (Fig. 3A, panel 1). Internal-
ization of OMVs in Int407 cells was confirmed by treatment with
different endocytic inhibitors prior to addition of OMVs. The
cholesterol-sequestering agent M␤CD inhibited the entry of V.
cholerae OMVs in Int407 cells (Fig. 3B, panels 5 and 6). This result
suggests that V. cholerae OMVs may be internalized into the cells
by cholesterol-enriched lipid rafts. M␤CD has also been reported
to inhibit clathrin-mediated endocytosis when applied at high
concentrations (33). Inhibition of vesicular endocytosis was fur-
ther confirmed by using nystatin, a cholesterol binding agent,
which disrupts lipid rafts but does not affect clathrin-mediated
endocytosis. Our results showed that nystatin also inhibits the
entry of V. cholerae OMVs in Int407 cells (Fig. 3B, panels 7 and 8).
Interestingly, dynasore (an inhibitor of the dynamin-dependent
endocytic pathway) failed to inhibit internalization of OMVs into
Int407 cells (Fig. 3B, panels 9 and 10). The inhibition of internal-
ization of C6709 OMVs is shown in Fig. 3B, which shows that
C6709 OMVs failed to enter Int407 cells upon treatment with
M␤CD and nystatin. The above results suggested that V. cholerae
OMVs are internalized into In407 cells by lipid raft-dependent
endocytosis.
To investigate OMV-mediated transport of HAP in Int407
cells, confocal microscopy was performed, using HAP antiserum
and FITC-conjugated secondary antibody. The merged images
showed colocalization of HAP and OMVs in Int407 cells when
treated with HAP-associated C6709 OMVs (Fig. 3A, panel 6),
whereas cells treated with CHA6.8 showed only OMV-specific red
fluorescence (Fig. 3A, panel 9). OMVs were labeled with VesC
antiserum and FITC-conjugated secondary antibody to assess
whether VesC is transported into cells. Our results with the colo-
calization assay clearly showed that VesC is transported into
Int407 cells through OMVs from hapA mutant strains CHA6.8
and CHA6.8 ⌬prtV (Fig. 3C, panels 6 and 9). Colocalization of
OMVs and VesC was not observed when cells were treated with
CHA6.8 ⌬prtV ⌬VC1649 OMVs. However, the merged image
showed adherence and internalization of OMVs in the absence of
VesC (Fig. 3C, panel 12), and this suggests that VesC is not respon-
sible for adherence of OMVs in Int407 cells. OMV-mediated
transport of VesC was further confirmed with OMVs from strain
Comp VC1649, which showed both OMV- and VesC-specific sig-
nals in the cells (Fig. 3C, panels 13 and 14).
OMV-associated proteases are biologically active. Studies
have shown that OMVs deliver cargo to the specific targets in an
active form (34). In the present study, we showed that both HAP
and VesC are transported into intestinal epithelial cells through
OMVs of their producing strains. HAP-associated C6709 OMVs
induced morphological changes of Int407 cells in a dose-depen-
dent manner when treated for 24 h (Fig. 4A, panel I). C6709
OMVs showed a cell-distending effect at a low concentration (20
␮g/ml), whereas they showed a cell-rounding effect similar to that
for purified HAP at a higher concentration (50 ␮g/ml) (Fig. 4A,
panel I). In contrast, such morphological changes were not ob-
served when cells were treated with OMVs from CHA6.8 and
other protease mutant strains. The proteolytic activity of HAP-
associated OMVs was studied on 79-kDa prohemolysin to con-
firm its biological activity. HAP has been reported to proteolyti-
cally cleave prohemolysin by digestion of the N-terminal
polypeptide chain to its 65-kDa mature form (7). Our results
showed that OMV-associated HAP digests 79-kDa prohemolysin
to produce 65-kDa and 50-kDa fragments, similar to the effect
observed with purified HAP (Fig. 4A, panel II). Our results con-
firmed that OMV-associated HAP is biologically active. In earlier
studies, we have reported that purified HAP and partially purified
VesC induce a hemorrhagic fluid response in the RIL assay (8, 10).
The biological activities of OMV-associated proteases were evalu-
ated in vivo by fluid accumulation assay in the ilea of adult mice.
OMVs from wild-type strain C6709 showed significant fluid ac-
cumulation (FA ratio, 0.20 Ϯ 0.03; n ϭ 5) (**, P Յ 0.01) compared
to the control (PBS treated) (Fig. 4B, panel I). Histopathological
studies of ileal tissues revealed widely dilated and ruptured villi
and the presence of inflammatory cells in the mucosa (magnifica-
tion, ϫ40) (Fig. 4B, panel IIB). Interestingly, hemorrhagic fluid
accumulation was observed in the intestinal segments when they
were treated with OMVs from CHA6.8 and CHA6.8 ⌬prtV. His-
topathological studies of the ileal tissues showed an altered villous
structure and mild hemorrhage in the submucosal membrane
when treated with CHA6.8 OMVs (Fig. 4B, panel IIC). However,
CHA6.8 ⌬prtV OMVs caused maximum damage of the ileal tis-
sues (Fig. 4B, panel IID). The ileal tissues showed grossly dis-
FIG 2 Localization of proteases in OMVs by proteinase K susceptibility assay.
OMVs from C6709 and CHA6.8 ⌬prtV were incubated at 37°C for 30 min with
or without 1 ␮g/ml proteinase K alone or with 1% SDS or 1 mM PMSF. Lane
1, control reaction; lane 2, sensitivity to extracellular protease; lane 3, inhibi-
tion of extracellular proteases; lane 4, protease sensitivity to luminal proteins;
lane 5, inhibition of protease activity by PMSF (control). OMVs were incu-
bated under the above-described conditions, precipitated with acetone, dried,
electrophoresed, and immunoblotted against HAP (A) or VesC (B) antiserum.
Mondal et al.

FIG 3 Colocalization of proteases and OMVs in the Int407 cell line. (A) Int407 cells were treated with OMVs from strain C6709 (panels 4, 5, and 6) or CHA6.8
(panels 7, 8, and 9) or with sterile PBS as a negative control (panels 1, 2, and 3), incubated with HAP and OMV antisera, and stained with TRITC (red)-conjugated
anti-mouse antibody (panels 1, 4, and 7). Red ﬂuorescence was detected in all OMVs except the negative control. HAP was detected using anti-HAP and
FITC-conjugated (green) anti-rabbit antibody in panel 5. Merged images are shown in panels 3, 6, and 9. The nucleus was stained with DAPI (4=,6=-diamidino-
2-phenylindole). (B) Inhibition of internalization of C6709 OMVs in Int407 cells treated with dynasore, methyl-␤-cyclodextrin (M␤CD), and nystatin prior to

rupted villi with hemorrhage in all layers of the mucosa. In con-
trast, ileal tissues treated with CHA6.8 ⌬prtV ⌬VC1649 OMVs
showed an almost normal villous structure with minimum hem-
orrhage in the mucosa and submucosa in histopathological stud-
ies (Fig. 4B, panel IIE). Fluid accumulation in the intestinal seg-
ments was also significantly reduced (FA ratio, 0.07 Ϯ 0.03; n ϭ
5). These results may suggest that OMV-associated VesC may be
responsible for the hemorrhagic fluid response in the mouse ileal
loop assay. The MIL assay with OMVs from Comp VC1649
showed restoration of hemorrhagic fluid in intestinal segments,
and histopathological studies showed mucosal damage and the
presence of inflammatory cells in the mucosa and submucosa (Fig.
4B, panel IIF). The presence of VesC in the OMVs may responsible
for the hemorrhagic fluid responses. However, it was reported
earlier that CT is responsible for fluid accumulation in the mouse
ileal loop assay (35). We measured the CT titers of V. cholerae
OMVs and culture supernatants by CT-bead ELISA to confirm
whether the OMV-mediated inflammatory responses is due to the
presence of CT in the OMVs. Both cell-free culture supernatants
and OMVs from V. cholerae strains grown in TSB showed almost
negligible CT titers compared to that of the positive-control strain
N16961 grown in Syncase medium (Fig. 4C) Therefore, the hem-
orrhagic response by OMVs in the MIL assay is not due to CT. The
above results confirmed that OMV-associated VesC is responsible
for hemorrhagic fluid accumulation in mouse ileal loops.
Host cell death induced by V. cholerae OMVs is dependent
on proteases. The cell death mechanisms induced by V. cholerae
OMVs were analyzed by flow cytometry. Flow cytometric analysis
with C6709 OMVs showed a dose-dependent apoptotic response
in Int407 cells (annexin Vϩ
/PIϪ
fraction). More than 50% cell
death was observed at 20 ␮g/ml of C6709 OMVs compared to the
untreated control. However, CHA6.8 OMVs induced greater cy-
totoxicity than C6709 OMVs, and cell death occurred by both
apoptosis and necrosis. Interestingly 30% reduced cell death was
observed when cell were treated with CHA6.8 ⌬prtV OMVs com-
pared to CHA6.8 OMVs. However, a 2-fold increase in the ne-
crotic cell population was observed compared to that with
CHA6.8 OMVs (Fig. 5C). CHA6.8 ⌬prtV ⌬VC1649 OMVs
showed reduced cytotoxicity compared to C6709, CHA6.8, and
CHA6.8 ⌬prtV OMVs. OMVs from the VC1649 complemented
strain restored the cytotoxicity in Int407 cells. These results clearly
demonstrated that VesC is responsible for necrotic damage of
Int407 cells (Fig. 5B and C).
VesC plays a role in intestinal colonization of V. cholerae in
the adult mouse model. Assessment of colonization of V. cholerae
strains in mouse intestinal epithelium showed significantly re-
duced bacterial colonization of the triple knockout mutant strain
CHA6.8 ⌬prtV ⌬VC1649 (CFU 3 ϫ 103
cells/g of intestine) com-
pared with the ⌬hapA ⌬prtV strain CHA6.8 ⌬prtV (CFU 8 ϫ 105
cells/g of intestine) (Fig. 6). The ⌬hapA strain CHA6.8 showed
increased colonization (CFU 1 ϫ 108
cells/g of intestine) com-
pared to wild-type strain C6709 (CFU 3 ϫ 107
cells/g of intestine).
There was a significant decrease in colonization in the CHA6.8
⌬prtV ⌬VC1649 strain compared to the CHA6.8 ⌬prtV strain.
Interestingly, when the VC1649 gene was complemented in
CHA6.8 ⌬prtV ⌬VC1649, the strain showed 3-fold-increased col-
onization. These results suggest that VesC may play a role in col-
onization in adult mice.
OMV-mediated IL-8 secretion from T84 cells is dependent
on VesC. OMVs isolated from C6709 and its protease mutant
Vibrio cholerae strains were incubated with T84 cells for 24 h, and
culture supernatants were taken and assayed for IL-8 expression
by ELISA and semiquantitative RT-PCR. Significant IL-8 mRNA
expression was observed when cells were treated with CHA6.8,
CHA6.8 ⌬prtV, and Comp VC1649, as visible IL-8-specific bands
were observed in agarose gel electrophoresis. OMVs from the
CHA6.8 ⌬prtV and Comp VC1649 strains induced maximum
mRNA expression (Fig. 7B). Protein expression in the same set of
experiments was studied by IL-8 ELISA to assess whether the IL-8
mRNA level is correlated at the protein level. The ELISA results
showed that OMVs from CHA6.8 induced more than a 2.5-fold
increase in IL-8 secretion compared to those from wild-type strain
C6709 and a ϳ10-fold increase compared to those from untreated
cells. Interestingly, IL-8 secretion was increased ϳ25-fold com-
pared to that in untreated cells when cells were treated with
CHA6.8 ⌬prtV OMVs in 24 h. This increased IL-8 secretion was
reduced 4-fold when cells were treated with OMVs from the VesC
mutant strain (Fig. 7A). Upon VesC complementation in the
hapA prtV VC1649 triple knockout mutant strain, IL-8 induction
by OMVs was significantly increased. These results suggest that
VesC may act as an inducer for OMV-mediated IL-8 secretion in
human intestinal epithelial cells.
DISCUSSION
Proteases produced by microorganisms play an important role in
virulence (3). Vibrio cholerae secretes hemagglutinin protease
(HAP), V. cholerae protease (PrtV), and serine proteases through
the type II secretion system (TIISS) (21, 36). Earlier we have re-
ported that purified HAP from a ctx-negative V. cholerae non-O1,
non-O139 strain and VesC from a ⌬hapA ⌬prtV Vibrio cholerae
strain may play a role in pathogenesis by inducing a hemorrhagic
response in the rabbit ileal loop (RIL) assay (8, 10). Our present
study demonstrated that HAP and VesC are also secreted in asso-
ciation with outer membrane vesicles (OMVs). Our results
showed that HAP is secreted in association with OMVs from wild-
type strain C6709, whereas the 59-kDa serine protease VesC is
secreted in association with OMVs from hapA mutant strains but
not from wild-type strain C6709. The absence of VesC in C6709
OMVs could be due to its proteolytic degradation in the presence
of HAP, as it has been reported to mask the secretion of other
proteases from V. cholerae (3, 10). Earlier studies have shown that
secretion of serine proteases is regulated by metalloproteases in
Vibrio species (3, 37). Syngkon et al. showed that 90% of the pro-
tease activity in strain C6709 is due to HAP, as only residual pro-
addition of OMVs. Cells with untreated OMVs were used as a negative control and cells without inhibition as a positive control. OMVs were labeled with
TRITC-conjugated antibody, and the nucleus was stained with DAPI. (C) Int407 cells were treated with C6709 OMVs (panels 1, 2, and 3), CHA6.8 OMVs (panels
4, 5, and 6), CHA6.8 ⌬prtV OMVs (panels 7, 8, and 9), CHA6.8 ⌬prtV⌬ VC1649 OMVs (panels 10, 11, and 12), and Comp VC1649 (panels 13, 14, and 15),
incubated with VesC and OMVs antisera, and stained with red TRITC-conjugated anti-mouse antibody (panels 1, 4, 7, 10, and 13). Red fluorescence were
detected in all OMVs. The presence of VesC was detected using anti-VesC and FITC-conjugated (green) anti-rabbit antibodies (panels 5, 8, and 14). Merged
images are shown in panels 3, 6, 9, 12, and 15. The nucleus was stained with DAPI.
Mondal et al.

FIG 4 Protease-associated OMVs are biologically active. (A) Biological activities of HAP-associated OMVs. Panel I, Int407 cells were treated with OMVs from
C6709 at 50 ␮g/ml and 20 ␮g/ml and OMVs from CHA6.8 for 24 h at 37°C. The higher concentration of C6709 OMVs showed a cell-rounding effect, whereas
the lower concentration showed a cell-distending effect. No morphological changes were observed in CHA6.8. Panel II, immunoblot analyses against hemolysin
antiserum with 10 ␮g purified hemolysin treated with HAP and C6709 OMVs and purified hemolysin as positive control. (B) Panel I, effect of OMVs of Vibrio
cholerae strains in the mouse ileal loop assay. Twenty micrograms (100 ␮g/ml) of OMVs from wild-type strain C6709 and its knockout derivatives was introduced
into ligated ileal loops of adult mice. The mice were sacrificed after 6 h, loops were excised, and fluid accumulation was calculated as the loop weight/length ratio.
Images of mouse ileal loops treated with V. cholerae OMVs are shown. The graph shows a summary of the data, represented as mean Ϯ SD (n ϭ 5 mice per group).
Variables were compared for significance using two-way analysis of variance and the Bonferroni test (*, P value between 0.01 and 0.05; **, P value between 0.01
and 0.001). Panel II, histopathological studies of mouse ileal loops. Twenty micrograms of OMV-treated ileal tissues was processed for histopathological analysis,
and photomicrographs were taken at a magnification of ϫ40. A, PBS-treated ileal tissues showed normal villi with mucosal structure; B, a magnified image of ileal
tissues treated with C6709 OMVs shows dilated and ruptured villi with accumulation of inflammatory cells in mucosal layers; C, CHA6.8 shows an altered villous
structure with mild hemorrhage in the submucosa; D, CHA 6.8 ⌬prtV OMV-treated ileal tissues show grossly disrupted villi with hemorrhage in all layers of the
mucosa; E, ileal tissues treated with OMVs of strain CHA6.8 ⌬prtV ⌬VC1649 show an almost-normal villous structure with minimum hemorrhage in the
mucosa, submucosa, and lamina propria; F, OMVs from the VC1649 complemented strain show accumulation of inflammatory cells and hemorrhage in mucosal
layers. (C) CT-beads ELISA results for OMVs from the C6709, CHA6.8, CHA6.8 ⌬prtV, CHA6.8⌬prtV ⌬VC1649, and Comp VC1649 strains. Culture super-
natant of strain N16961 was used as a positive control.

tease activity was detected in hapA and prtV mutant strains (10).
Sikora et al. reported the presence of several serine proteases, i.e.,
VesA, VesB, and VesC, in V. cholerae O1 El Tor strain N16961, in
which significantly less HAP is produced due to a frameshift mu-
tation in HapR (38). However, Altindis et al. showed the presence
of VesC in the OMVs of V. cholerae wild-type strain C6706 when
grown under conditions which activate TCP (22). HAP was not
detected in the OMVs of the wild-type strain under these condi-
tions, as production of HAP is coordinated in a fashion reciprocal
to that for cholera toxin and TCP (39). Secretion of HAP from
OMVs of V. cholerae is influenced by serine protease DegP, and
the degP mutant strains have been shown to secrete HAP and 8
other proteins from outer membrane vesicles (22).
Immunoblotting results showed the presence of mature 45-
kDa HAP in C6709 OMVs instead of its processed 35-kDa form
secreted through the TIISS. The mature 45-kDa form of HAP was
FIG 6 Comparative analysis of colonization of Vibrio cholerae strain C6709
and its isogenic knockout mutants in mouse intestine. A summary of the data,
represented as mean Ϯ SD (n ϭ 10 mice per group), is shown. Variables were
compared for significance using two-way analysis of variance and the Bonfer-
roni test (*, P value between 0.01 and 0.05; **, P value between 0.01 to 0.001).
FIG 5 Host cell death induced by OMVs from V. cholerae strains. (A) Flow cytometric analysis of cell death induced by V. cholerae OMVs. The graphical displays
show data from a representative experiment. In each display the lower right quadrant represents apoptotic cells (annexin Vϩ
/PIϪ
), and the upper left and right
quadrants represent necrotic cells (annexin Vϩ
/PIϩ
and annexin VϪ
/PIϩ
fractions). Upper panel, untreated Int407 cells and Int407 cells treated with 20 ␮g/ml
and 40 ␮g/ml of OMVs from V. cholerae strain C6709. Lower panel, Int407 cells treated with 20 ␮g/ml and 40 ␮g/ml OMVs from isogenic protease mutants of
the C6709 and Comp VC1649 strains. (B) Results from three independent flow cytometry experiments. Data are represented as mean (ϮSD) percentage of dead
cells after treatment with 20 and 40 ␮g/ml of OMVs from V. cholerae strains (n ϭ 3). (C) Fractions of apoptotic and necrotic cells of the same experiment. Data
are represented as mean (ϮSD) percentages of apoptotic and necrotic cells (n ϭ 3).
Mondal et al.

purified earlier by Ghosh et al. in the presence of the metallopro-
tease inhibitor EDTA, whereas in its absence the processed 35-kDa
form is purified from culture supernatant (8). The protease un-
dergoes several steps of processing, including cleavage of the signal
peptide and further processing of the N terminus and C terminus
to generate the 35-kDa protein (4). The presence of the 45-kDa
form of HAP within the C6709 OMVs could be due to its lu-
minal localization within the vesicles. Earlier studies have
shown that luminal localization of proteins within OMVs pro-
tects them from proteolytic digestions (40). Therefore, it may
be suggested that the 45-kDa HAP is protected within the
OMVs from further processing.
OMVs have been shown to deliver different proteins and tox-
ins into host cells in an active form (40). Several studies have
shown that toxins such as CT, RTX, VCC, and metalloprotease
PrtV in V. cholerae are transported to host cells through OMVs in
a biologically active form (19, 20, 41). Our present study showed
that both HAP- and VesC-associated OMVs are internalized into
human intestinal epithelial cells (Int407) in a protease-indepen-
dent manner. Confocal microscopic images revealed that these
OMVs may be internalized into host cells through lipid rafts, as
vesicular endocytosis was completely inhibited by M␤CD and
nystatin.
The proteases in OMVs are transported in biologically active
forms. OMV-associated HAP induces dose-dependent morpho-
logical changes in Int407 cells similar to those shown by purified
HAP in our earlier study (8). HAP has been shown to proteolyti-
cally activate El Tor hemolysin/cytolysin by nicking at its N-ter-
minal peptide to generate the 65-kDa mature form (7). Our results
showed that OMV-associated HAP also digests the 79-kDa pro-
hemolysin to generate 65-kDa and 50-kDa mature forms. The
50-kDa mature hemolysin is a C-terminally processed, identically
truncated monomer of the 65-kDa mature hemolysin reported by
Ekigai et al. in V. cholerae O1 strains (42).
The roles of OMV-associated proteases in pathogenesis were
studied in the mouse ileal loop (MIL) model. This is the first study
to show fluid accumulation in mouse ileal loops caused by V.
cholerae OMV-associated proteases. HAP-associated OMVs
showed an enterotoxic response in the MIL assay. VesC-associated
CHA6.8 ⌬prtV OMVs showed a visible hemorrhagic fluid re-
sponse in MILs. Histopathological analysis of the mice ileum
showed grossly disrupted villi with hemorrhage in all layers of the
mucosa, which was significantly reduced upon treatment with
CHA6. 8⌬prtV ⌬VC1649 OMVs. These effects were restored with
OMVs from the VC1649 complemented strain. A similar hemor-
rhagic fluid response was observed with partially purified VesC
and culture supernatants from CHA6.8 ⌬prtV in rabbit ileal loop
(RIL) assay by Syngkon et al. They showed significant reduction of
hemorrhagic fluid when inhibited with PMSF (10). This hemor-
rhagic fluid accumulation was not due to cholera toxin, as CT-
FIG 7 IL-8 induction by OMVs of V. cholerae O1 strains in the T84 cell line. (A) Comparative analysis of IL-8 secretion from T84 cells coincubated with OMVs
from V. cholerae O1 El Tor strain C6709, its isogenic protease mutant strains CHA6.8, CHA6.8 ⌬prtV, and CHA6.8 ⌬prtV ⌬VC1649, and CHA6.8 ⌬prtV
⌬VC1649::VC1649 (Comp VC1649) determined by ELISA. Data represent mean Ϯ SD from three independent experiments performed under similar condi-
tions. Variables were compared for significance using two-way analysis of variance and the Bonferroni test (*, P value between 0.01 and0.05; **, P value between
0.01 and 0.001). (B) IL-8 production from T84 cells was analyzed at the mRNA and protein levels by RT-PCR and resolved in a 2% agarose gel. IL-8 expression
in T84 cells treated with OMVs from O1 El Tor strain C6709 and its protease knockout mutant strains CHA6.8, CHA6.8 ⌬prtV, CHA6.8 ⌬prtV ⌬VC1649, and
Comp VC1649 is displayed in lanes 2 to 5, respectively, and the untreated control is in lane 1. Densitometric quantification in densitometric units (DU) for IL-8
after normalization to GAPDH is shown below the agarose gel.

bead ELISA showed the absence of CT in V. cholerae OMVs (Fig.
4C). The OMV-free culture supernatants also showed the absence
of CT in bead ELISA (data not shown). This may be due to differ-
ent conditions for production of CT and HAP. Benitez et al.
showed that growth conditions that favor production of HAP
downregulate CT and TCP and vice versa (43).
OMV-associated proteases of V. cholerae induced cytotoxic ef-
fects in Int407 cells in a dose-dependent manner. C6709 OMVs
induced an apoptotic response in Int407 cells. Thay et al. have
shown that membrane-derived vesicles from Staphylococcus au-
reus induce apoptosis in a dose-dependent manner by an ␣-toxin-
dependent mechanism (29). However, the apoptotic cell popula-
tion was not significantly reduced when treated with CHA6.8
OMVs. CHA6.8 OMVs induced a greater cytotoxic response with
an increase in necrotic cells compared to HAP-associated C6709
OMVs. Studies have shown that there is an increased virulence in
hapA-deleted V. cholerae strains. Benitez et al. showed that inac-
tivation of hapA results in increased intestinal colonization and
adherence of V. cholerae in human intestinal epithelial cells (44).
Zhou et al. also reported that hapA-defective cholera vaccine can-
didate strains showed reactogenicity. Culture supernatants of
these strains can stimulate IL-8 secretion from T-84 cells (45).
Therefore, it may be suggested that the presence of metallopro-
tease PrtV in CHA6.8 OMVs may be responsible for increased
OMV-mediated cytotoxicity. However, earlier studies have shown
that PrtV modulates IL-8 secretion from T84 cells (46). Interest-
ingly, when cells were treated with CHA6.8 ⌬prtV, a shift from an
apoptotic population to a necrotic population was observed. In
CHA6.8 ⌬prtV ⌬VC1649 OMVs, the cytotoxicity was significantly
reduced, and increased cytotoxicity in Int407 cells was restored
with OMVs from the VC1649 complemented strain. These results
are very similar to the effect observed with OMVs on MIL assay.
Earlier studies on the pathogenicity of OMVs in bacterial in-
fections have shown the role of lipopolysaccharide (LPS)- and
OMV pathogen-associated molecular patterns (47, 48). LPS in the
OMVs is sensed by the Toll-like receptor 4 (TLR4) complex to
trigger inflammatory responses (49). OMVs isolated from Salmo-
nella enterica serovar Typhimurium activate macrophages and
dendritic cells to increase levels of surface major histocompatibil-
ity complex (MHC) class II expression as well as the production of
the proinflammatory mediators tumor necrosis factor alpha
(TNF-␣) and interleukin-12 (IL-12) (50). Helicobacter pylori and
Pseudomonas aeruginosa OMVs show a proinflammatory re-
sponse by IL-8 secretion (51, 52). IL-8 is a major proinflammatory
cytokine which act as a potent chemoattractant for polymorpho-
nuclear leukocytes (PMN) and recruits them into infected sites of
the epithelial layer, which results in opening of epithelial tight
junctions (53). The IL-8 response of Vibrio cholerae OMVs was
shown by Chatterjee and Chaudhuri. They showed that OMVs
from classical V. cholerae strain O395 induce IL-8 secretion from
intestinal epithelial cells, which activates dendritic cells to pro-
mote T-cell polarization (54). However, our results showed that
HAP-associated OMVs from El Tor strain C6709 failed to induce
significant IL-8 secretion from T84 cells. Earlier studies have also
shown that HAP does not induce IL-8 secretion from intestinal
epithelial cells (45). OMVs from ⌬hapA strain CHA6.8 showed
ϳ10-fold-increased IL-8 secretion compared to untreated con-
trols, whereas IL-8 secretion was increased ϳ25-fold upon treat-
ment with CHA6.8 ⌬prtV OMVs. The greater IL-8 response by
OMVs from the ⌬hapA ⌬prtV double mutant strain could be due
to the absence of PrtV, as it has been reported to reduce IL-8
secretion from intestinal epithelial cells (46). This increased IL-8
secretion by CHA6.8 ⌬prtV was significantly reduced upon treat-
ment with OMVs from the VesC mutant strain. Interestingly,
OMVs from Comp VC1649 showed significant induction of IL-8
secretion. These results show that VesC within the V. cholerae
OMVs is responsible for the increased IL-8 response in T84 cells.
Our results revealed 3-fold-increased colonization of the
CHA6.8 ⌬prtV strain compared to the VesC-deleted
CHA6.8⌬prtV⌬VC1649 strain in adult mice. Interestingly in-
creased colonization of V. cholerae was restored with the VC1649
complemented strain. Earlier, Silva et al. (59) also reported an
increased colonization of hapA mutant strains in infant mouse
model. However, Sikora et al. reported that VesC is not responsi-
ble for colonization of V. cholerae in infant mouse model (38). Our
results showed that VesC may play an important role in coloniza-
tion in adult mice.
OMVs have recently been used to develop acellular vaccines for
several diseases related to Gram-negative bacteria. The most suc-
cessful use of OMVs as a vaccine candidate has been against sero-
group B Neisseria meningitides infection, for which over 55 million
doses have been administered to date against serogroup B infec-
tions (55). Schild et al. showed that OMVs from V. cholerae can
generate a protective immune response against cholera infection
in the adult mouse model (25). However, our results suggest that
the presence of proteases in the OMVs may affect the use of OMVs
as a vaccine candidate for cholera.
In conclusion, we showed that the proteases HAP and VesC are
secreted in association with V. cholerae OMVs in an active form.
OMV-associated VesC induces a hemorrhagic response in the
MIL assay and a proinflammatory response in human cultured
intestinal epithelial cells, and VesC may play an important role in
colonization in the adult mouse model.
ACKNOWLEDGMENTS
This work was supported by intramural funding from the Indian Council
of Medical Research. A.M. was supported by a fellowship from the Indian
Council of Medical Research.
We thank Somnath Chatterjee, confocal microscope operator, NICED
central facility (Carl Zeiss, India), for technical help with confocal micro-
scopic studies, Somdatta Chatterjee, Division of Bacteriology, NICED, for
crucial assistance with VC1649 complementation, and Amarshi Mukher-
jee, Division of Biochemistry, NICED, for providing purified hemolysin.
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